The proposed research aims to establish the feasibility of utilizing the malignant tumor of planarians, which is induced by chemical cocarcinogens, as an in vivo model system for studies on the mechanisms of tumor initiation and promotion. The pathogenesis of this inducible malignant tumor models several important features of a neoplastic stem cell disease similar to those of the hematopoietic cell lines of mammals, including humans. The precise sequence and timing of cocarcinogen treatment for tumor expression will be determined by further assessing the mutagenic effects of cadmium as an initiator and epigenetic influences of 12- O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Possible protection against mutagenic damages (alterations of DNA replication or DNA repair systems) induced by cadmium will be tested by pre- or post-treatment of metallothionein. To test whether TPA is acting through protein kinase C in planarian stem cells as in mammalian cells, the capacity of various tumor promoting and non-promoting phorbol analogs will be examined for their capability to displace (3H)-PDBu (phorbol-12,13-dibutyrate) binding. Furthermore, to test whether inhibitors of protein kinase C can competitively antagonize both PDBu binding and tumorigenicity, planarians will be treated with sphingosine, during the exposure to cadmium and TPA. It will be determined by the tissue grafting whether the stem cells located in the postpharyngeal region are more susceptable to cadmium and TPA than those located in the other region of the body. In order to verify the malignancy of the cocarcinogen-induced planarian tumor, the transplantability test will be done by serial grafting of the same tumor and/or by injection of isolated, transformed stem cells into normal planarians. Using the cell freezing and thawing techniques, the isolated, transformed stem cells after 3 months of storage in the frozen state will be tested for their transplantability. The pathogenetical lesions will be examined by electron microscopy. These studies should aid in understanding the mechanisms of tumor initiation and promotion, and the fundamental similarity of tumorigenesis in invertebrate cells to vertebrate cells, and will extend the usefulness of a simple in vivo model system for further research.